Microscopy has an important part in life sciences. Biological samples can be present on a number of different sample carriers, for example between slide and cover glass, in Petri dishes or microtiter plates. They can be still alive or fixed, non-dyed or dyed. In general, they can be observed in transmitted light and reflected light.
In case of transmitted light illumination, the light emitted by a light source irradiates the sample and is collected by the lens and directed onto a detector. Part of the light is absorbed by the sample, diffused or refracted, which manifests as a contrast in intensity on the detector. If the sample does not absorb any or hardly any light, which is often the case for thin biological specimens, particularly if they are non-dyed, the phase structure of the sample can be made visible by means of a number of contrast methods (phase contrast, differential interference contrast—DIC, etc.). Admittedly, all these methods have in common that they can represent the shape of biological samples, such as cells, for example, as well as some of their components (cell nucleus, et al.); functional statements, however, are frequently impossible.
Fluorescence dyes, however, allow dyeing of specifically targeted cell components. The dyes are excited with light of suitable wavelength for the purpose of imaging, which is ordinarily implemented by reflected light illumination, with the illumination light being focused through the lens onto the observed sample area.
The fluorescence signal—red-shiftet with respect to the excitation light—is acquired by the same lens, separated from the excitation light by means of a dichroite and matching filters, and directed to the camera or the oculars. Statements regarding the functions and functional changes in the cells can be derived from the fluorescence images. In doing so, however, information on the cell shape in its entirety is often missing.
It is therefore advantageous to combine transmitted light and fluorescence images with one another because the corresponding images supplement each other regarding their statements. Therefore typically are required two illumination systems, which are separated from one another and which are being operated sequentially. Thus, a transmitted light image of a sample can be acquired, first, for example, and subsequently a fluorescence image is acquired.
To save time, transmitted light and fluorescence images can also be implemented in parallel. Therefore are required two cameras on the one hand. On the other hand, the wavelength of the transmitted light must be a different one than that of the fluorescence signal so that both signals can be separated from one another by means of a dichroic mirror. Therefore, the time gain is bought with a significantly more complex structure.
It would be advantageous to combine the transmitted light and fluorescence images with one another without having to accept time loss or significantly more complex structures.
U.S. Pat. No. 4,515,445 for example, discloses a method, in which a reflected light illumination is directed onto the sample. That part of the light, which is transmitted through the sample, is reflected back through a mirror in a plane conjugate to the sample plane and, in doing so, functions as transmitted light during the second round of sampling. This results in an image, which is created both by parts of transmitted light and by light, which is reflected by the sample. This method, however, is not suitable for fluorescence images, because, admittedly, a dichroic mirror can be introduced into the optical path, which separates the light emitted by the fluorophores from the excitation light, but would also filter out the transmitted light during the second round of sampling. The advantage of such method would only consist in directing the light, which was not utilized to excite the fluorophores during the first round of sampling, to the sample, once again, thereby increasing the excitation intensity. The result, however, is a standard fluorescence image.
A different solution is described in Ding et al., Optics Express 20, 14100-14108 (2012). The sample is illuminated point for point in a laser scanning configuration, albeit obliquely, in each case. A fluorescent layer is located behind the sample, which layer is excited by the laser light, which is passing the sample, and in turn sends out a fluorescence signal. This signal, in turn, serves as transmitted light, if it is caught by the lens through the sample and directed in the direction of the detector. As a result of the excitation light being incident obliquely to the sample, it also excites a region behind the sample in the fluorophore layer, which is positioned offset with respect to the optical axis. Therefore, the transmitted light sent out from there, also passes the scanned sample point at just that angle. This results in an image, which is similar to that of a classical oblique illumination. If the sample itself is fluorescent, as well, the fluorescence signal of the sample is added to the image. Such structure, however, is extremely sophisticated and expensive.